Family of proteins plays key role in cellular pump dynamics

Case Western Reserve University scientists have discovered how a family of proteins — cation diffusion facilitators (CDFs) — regulates an important cellular cycle where a cell's energy generated is converted to necessary cellular functions. The finding has the potential to inform future research aimed at identifying ways to ensure the process works as designed and, if successful, could lead to significant breakthroughs in the treatment of Parkinson's, chronic liver disease and heart disease.

The results of this research were posted online June 22 by the journal Nature and will be published in the print edition at a later date.

"CDF is a major protein family type found in all forms of life," said senior author Mark R. Chance, PhD, the Charles W. and Iona A. Mathias Professor of Cancer Research, Case Western Reserve University School of Medicine. "Mutations or altered regulation of human CDFs modify the concentrations of metal ions critical to cell function and are associated with key human diseases, including those affecting endocrine, neurologic, hepatic and cardiovascular systems."

To understand how the cell cycle works, envision a gate to human cells that controls the flow of substances necessary to maintain cell survival. When and how that gate opens and closes is critical to fundamental cellular functions, and in turn, to human health. CDFs ensure the gate's seamless operation by controlling the flow of metal ions as energy is cycled. In this investigation, Case Western Reserve scientists sought to understand the intricate details of CDF molecular function and mechanisms of transport.

Chance and his colleagues studied a form of CDF found in bacteria where the protein YiiP functions like a motor, using energy in the form of a gradient of protons (hydrogen atoms) to pump zinc ions out of cells.

While zinc is pushed successfully out of the cell, a flow of protons is pulled into it. A perfectly functioning zinc-proton flow cycle, then, brings in protons, which the YiiP protein converts into conformational changes in the protein structure. Those changes, in turn, send zinc out of the cell. If the CDF-regulated gate controlling the zinc-proton cycle malfunctions, a range of diseases can result.

To visualize the zinc-proton cycle, Case Western Reserve scientists used sophisticated dynamic imaging technology — the cellular cycle operates on time scales comparable to the blink of the eye. Dynamic imaging involved a labeling system — x-ray-mediated hydroxyl radical footprinting — that recognizes water molecules in transmembrane proteins. The scientists also used mass spectrometry, a powerful atom-and-molecule recognition technology, to study the labeled proteins. These technologies allowed investigators to watch the YiiP protein in real time as it took up zinc atoms and rearranged its structure cycle through a pumping sequence.

"Membrane proteins (including CDF) are some of the most important cellular drug targets, including G-protein coupled receptors (GPCR), which represent 50 percent of the non-antibiotic drug market." Chance said.

GPCRs are protein molecules that sense chemical signals outside the cell and then activate cellular responses to these signals. Chance and his colleagues have studied GPCR structure and dynamics using innovative mass spectrometry-based technology. In this more recent work on CDFs, a dynamic picture of the membrane protein has emerged. It is a complex, but explainable, machine that uses a widely available form of energy, the proton gradient, to carry out cellular functions.

"We have now produced high-resolution pictures of signal transmission and ion transport mechanisms for a range of ion channels and GPCRs," Chance said. "Our work in CDFs is a visible example of the power of these new technologies to solve important problems in the membrane protein field. We must continue to examine CDFs to understand their mechanisms of action, especially in the context of drug effects on the biochemical mechanisms of action."

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Collaborating with Chance on this investigation was senior author Dax Fu, PhD, Johns Hopkins University School of Medicine, Baltimore. Additional researchers included Sayan Gupta, PhD, Case Western Reserve University School of Medicine (lead author); Jin Chai, Brookhaven National Laboratory, Upton, N.Y.; Jie Cheng, MD, PhD, Johns Hopkins University School of Medicine; and Rhijuta D'Mello, Case Western Reserve University School of Medicine.

This research is supported in part by the National Institutes of Health (NIH) under grant R01 GM065137; Office of Basic Energy Sciences, U.S. Department of Energy grant DOE KC0304000; and NIH's National Institute for Biomedical Imaging and Bioengineering under grants P30-EB-09998 and R01-EB-09688. The National Synchrotron Light Source at Brookhaven National Laboratory is supported by the U.S. Department of Energy under contract DE-AC02-98CH10886.

About Case Western Reserve University School of Medicine

Founded in 1843, Case Western Reserve University School of Medicine is the largest medical research institution in Ohio and is among the nation's top medical schools for research funding from the National Institutes of Health. The School of Medicine is recognized throughout the international medical community for outstanding achievements in teaching. The School's innovative and pioneering Western Reserve2 curriculum interweaves four themes--research and scholarship, clinical mastery, leadership, and civic professionalism--to prepare students for the practice of evidence-based medicine in the rapidly changing health care environment of the 21st century. Nine Nobel Laureates have been affiliated with the School of Medicine.

Annually, the School of Medicine trains more than 800 MD and MD/PhD students and ranks in the top 25 among U.S. research-oriented medical schools as designated by U.S. News & World Report's "Guide to Graduate Education."

The School of Medicine's primary affiliate is University Hospitals Case Medical Center and is additionally affiliated with MetroHealth Medical Center, the Louis Stokes Cleveland Department of Veterans Affairs Medical Center, and the Cleveland Clinic, with which it established the Cleveland Clinic Lerner College of Medicine of Case Western Reserve University in 2002. http://casemed.case.edu